Objective: To establish an HPLC method for quantitative determination of fidaxomicin in fermentation broth. Methods: The separation was carried out on the Agilent 5 TC-C18 (250 mm&#215;4.6 mm, 5 μm)column at the detection wavelength of 228 nm and the column temperature was set at 40℃. The mobile phase consisted of methanol-0.1% formic acid solution at a flow rate of 1.0 mL&#183;min-1. Results: The mass concentration of fidaxomicin and peak area showed excellent linearity within the range of 6.59-79.08 μg&#183;mL-1, and the correlation coefficient was 0.999 9 (n=7). The average recovery (n=9) was 98.6% (RSD=0.91%). The precision, reproducibility and stability were satisfactory with RSD of less than 2%. The contents of fidaxomicin in three batches of fermentation broths were 59.4, 52.5 and 53.6 μg&#183;mL-1, respectively. Conclusion: The developed method is suitable for strain screening and fermentation control of fidaxomicin.